Prolyl oligopeptidase (EC 3.4.21.26) (POP), also known as prolyl endopeptidase (PREP), is a serine protease that catalyses the hydrolysis of peptides at the C-terminal side of L-proline residues. It is widely distributed in mammals and can be purified from various organs, including the brain.
The enzyme plays an important role in the breakdown of proline-containing neuropeptides related to learning and memory functions (Wilk S et al., Life Sci. 1983; 33:2149-57; O'Leary R M, O'Connor B. J. Neurochem. 1995; 65:953-63).
The effects of prolyl oligopeptidase inhibition have been tested in the treatment of cognitive deficits related to neurodegenerative processes. Parkinson's disease was generated in monkeys by treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that produces depletion of substance P. Subsequent treatment with S-17092, a potent POP inhibitor, increased the performance of cognitive tasks (Schneider J S et al., Neuropsychopharmacology 2002; 26(2):176-82). It has also been found that POP inhibition prevents the oligomerization of α-synuclein ex vivo [Myöhänen T T et al., Br. J. Pharmacol. 2012; 166(3):1097-113]. In the case of Alzheimer's disease (AD), several in vivo experiments in animal models showed that POP inhibition led to neuroprotective and cognition-enhancing effects (Kato A et al., J. Pharmacol. Exp. Ther. 1997; 283(1):328-35 and Toide K et al., Rev. Neurosci. 1998; 9(1):17-29). Neuroprotective effects were originally observed by Katsube's group, when cortical and cerebellar granule cells were prevented from age-induced apoptosis by treatment with the POP inhibitor ONO-1603 (Katsube N et al., J. Pharmacol. Exp. Ther. 1999:288(1):6-13).
Clinical trials with POP inhibitors in the treatment of cognitive deficits have been performed only in a few cases. In a phase I clinical study Morain's group (Morain P et al., Br. J. Clin. Pharmacol. 2000; 50(4):350-9) found that S 17092, a new orally active prolyl endopeptidase inhibitor, showed cognition-enhancing properties in healthy elderly subjects and a clear dose-dependency; moreover, no adverse effects were detected. Later studies suggested additional slight mood-stabilizing properties for this compound (Morain P et al., Neuropsychobiology 2007; 55(3-4):176-83).
Prolyl oligopeptidase activity has been reported to be altered (post-mortem) in several neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease, Huntington's disease and multiple sclerosis (MS) (Mantle D et al., Clin. Chim. Acta 1996; 249(1-2):129-39).
There is also a substantial amount of evidence pointing to a role for neuroinflammation in the pathogenesis of neurodegenerative diseases such as AD, MS and Parkinson's disease (Hirsch E C et al., Lancet Neurol. 2009; 8(4):382-97, Philips T et al., Lancet Neurol. 2011; 10(3):253-63). POP has been considered to be the main enzyme implicated in the release of an anti-inflammatory tetrapeptide Ac-SDKP from Tβ4 in the brain (Yang F et al., Hypertension 2004; 43(2):229-36, Nolte W M et al., Biochemistry 2009; 48(50):11971-81). This suggests that the inhibition of POP may help reduce neuroinflammation and consequently POP inhibitors may be useful in the treatment of neurodegenerative diseases with an inflammatory component such as Alzheimer's and Parkinson's disease and in particular help improve the cognitive disorders associated with these diseases.
Senile plaques spreading over the cortical brain areas are typical neuropathological hallmarks of AD. The main protein component of these plaques is amyloid β-peptide (Aβ). Deposition of Aβ triggers neuronal dysfunction and death in the brain. This peptide derives from the β-amyloid precursor protein (APP). Under normal conditions, APP is cleaved by α-secretase to generate soluble APPα which precludes Aβ generation.
Interestingly, POP inhibition increases intracellular IP3 levels, which may contribute to the stimulation of APPα production, which would in turn decrease Aβ generation.
Additionally, Rossner (Rossner S et al., Neurochem. Res. 2005; 30(6-7):695-702) found less POP immunoreactive neurons in brain structures of AD patients affected by Aβ plaques.
Additionally it seems that substance P can suppress neurotoxic action of β-amyloid protein (Kowall N W et al., Proc. Natl. Acad. Sci. USA 1991; 88(16):7247-51). Prolyl oligopeptidase inhibitors inhibit the metabolism of substance P helping to sustain levels of substance P that may suppress the neurotoxic action of β-amyloid protein.
In view of the above mentioned effects, it is thought that prolyl oligopeptidase inhibitors may be useful drugs for the treatment of Alzheimer's disease helping to improve the cognitive disorders associated with the disease.
Prolyl oligopeptidase has also been associated with several factors that might be relevant to multiple sclerosis (MS). For instance, POP is involved in the regulation of microglia toxicity (Klegeris A et al., Glia 2008; 56(6):675-85). Indeed, a recent report established a direct connection between POP and MS; the plasma POP activities of patients with RR-MS were significantly reduced (Tenorio-Laranga J et al., J Neuroinflammation 2010; 7:23). Interestingly, the reduction correlated with the severity of disease symptoms, but not with patient age. Instead, an inverse correlation between POP activity and age was observed in healthy controls, and in elderly controls the levels were comparable to those found in MS patients.
The neuropathological hallmark of Parkinson's disease is the progressive degeneration of melanised dopaminergic neurons in substantia nigra pars compacta together with intracellular inclusions known as Lewy bodies. A major component of the Lewy bodies is a 140 amino acid protein, α-synuclein. Under certain conditions, α-synuclein monomers interact to form prefibriliar aggregates or protofibrils, which can create cytotoxic insoluble fibrils. These fibrils cannot be degraded by the proteasome, and they impair the function of this intracellular proteolytic system. This leads to an accumulation of α-synuclein protofibrils (and other proteins that are degraded by the proteasome) in the cytosol (Bennett M C, Pharmacol. Ther. 2005; 105(3):311-31) and as a consequence, α-synuclein protofibrils are increased in brains of Parkinson's disease patients. These fibrils have been associated with neurotoxicity in α-synuclein overexpressing cells and mouse models (Masliah E et al., Science 2000; 287(5456): 1265-9; Gosavi N et al., J. Biol. Chem. 2002; 277(50):48984-92). Abnormal accumulation of misfolded α-synuclein may lead to mitochondrial changes which can promote oxidative stress and evoke cell death (Hsu L J et al., Am. J. Pathol. 2000; 157(2):401-10). Furthermore, three point mutations (A53T, A30P or E48K) in the α-synuclein gene are known to be involved in the pathogenesis of familial form of Parkinson's disease (Polymeropoulos M H et al., Science 1997; 276(5321):2045-7; Zarranz J J et al., Ann. Neurol. 2004; 55(2):164-73).
It has been shown in vitro that the aggregation rate of α-synuclein was enhanced when the protein was incubated with a clone of wild-type porcine POP, and this enhancement depended upon the POP concentration (Brandt I et al., Peptides 2008; 29(9):1472-8). Moreover, a mutated variant without POP activity (S544A) did not accelerate the aggregation rate.
Enhanced aggregation could also be prevented by the addition of POP inhibitors, suggesting that the effect was dependent on the POP enzymatic activity. Recent evidence has suggested that POP inhibitors can block the increased α-synuclein aggregation induced by oxidative stress in human α-synuclein overexpressing neuroblastoma SH-SY5Y cells Myöhänen T T et al., Br J. Pharmacol 2012; 166(3):1097-113. POP colocalizes with α-synuclein in SH-SY5Y cells, and this colocalization disappears after incubation with POP inhibitors, pointing to an interaction between POP and α-synuclein. A 5-day treatment with a POP inhibitor reduced the amount of soluble α-synuclein in the brains of a A30P α-synuclein transgenic mice.
Thus, inhibition of brain POP activity could prevent α-synuclein aggregation and thus, prevent the formation of the cytotoxic protofibrils present in the Lewy bodies. Therefore, POP inhibitors could potentially have therapeutical value in the treatment of neurodegenerative disorders where accelerated α-synuclein aggregation has been described.
Compounds capable of inhibiting POP are effective for preventing experimental amnesia induced by scopolamine in rats, inferring that POP inhibitors have functions in the alleviation of mnemonic dysfunctions (Yoshimoto T et al., J. Pharmacobiodyn. 1987; 10:730-5).
The effect of subchronic administration of rosmarinic acid, a non-competitive POP inhibitor (with a relatively high IC50 value of 63.7 μM), was tested in the Morris water maze in rats, and an enhancement in spatial memory was reported (Park D H et al., Fitoterapia 2910; 81(6):644-8).
It has been found that patients with bipolar disorder have high levels of activity of the POP in serum. In recent years, POP has gained importance as a target for the treatment of this disease, especially due to his involvement in the metabolism of inositol-1,4,5-P3 (IP3). IP3 is a key molecule in the transduction of the signal in the cascade of neuropeptides. Through the binding to specific receptors, neuropeptides induce an increase of IP3, which binds to its receptor on the membrane of the endoplasmic reticulum and induces the release of Ca2+, which is believed to play a crucial role in learning and memory. Recent findings have shown that the POP modulates the concentration of IP3 (Komatsu Y J. Neurosci. 1996; 16:6342-52). Thus it is known that a disruption of the gene of the POP in the eukaryotic Dictyostelium discoideum induces resistance to lithium via elevation of IP3 (Schulz I et al., Eur. J. Biochem. 2002; 289:5813-20), and also reduced the proteolytic activity of POP, which is responsible for the high concentration IP3 in glioma cells antisense human for POP. This effect is also observed when these cells are treated with specific POP inhibitors (Williams R S et al., EMBO J. 1999; 18:2734-45).
The IP3 signaling pathway is involved in the action of several drugs therapeutic mood stabilizers (lithium, carbamazepine and valproic acid) and defects in the mechanisms that regulate the IP3 signaling may cause bipolar disorder. Moreover, the mood stabilizer drug that is commonly used to treat bipolar disorder, valproic acid, directly inhibits the activity of recombinant POP (Cheng L et al., Mol. Cell. Neurosci. 2005; 29:155-61). In summary, there is strong evidence that POP inhibitors are useful in the prevention and/or treatment of bipolar affective disorder in mammals. Thus, to provide novel inhibitors of POP is interesting in the therapy of this disorder or disease.
In summary, the effects of several POP inhibitors in various cognitive tasks have been characterized, and there is some kind of consensus that POP inhibitors have positive effects on learning and memory (Morain P et al., CNS Drug. Rev. 2002; 8(1):31-52; Shinoda M et al., Eur. J. Pharmacol. 1996; 305(1-3):31-8; Marighetto A et al., Learn. Mem. 2000; 7(3):159-69; Toide K et al., Pharmacol. Biochem. Behav. 1997; 56(3):427-34; Schneider J S et al., Neuropsychopharmacology 2002; 26(2):176-82).
Several patents and patent applications disclose POP inhibitors: WO 2008/077978 A1, WO 2005/027934 A1, JP 2011-037874 A2, WO 2005/002624 A1, WO 2004/060862 A2, WO 03/04468 A1; DE 196 03 510 A1, US 2006/0100253 A1 and U.S. Pat. No. 6,159,938 A, but only a few compounds have undergone in vivo studies (JTP-4819, S 17092, Z-321, ONO-1603, Y-29794, ZTTA, Z-Pro-Prolinal, and KYP-2047), only the first three in the list have entered clinical trials and none of them has reached the market place.
In spite of the existence of POP inhibitors, there is still a need in the art to provide alternative compounds with a high affinity to POP and a good capacity to cross the blood-brain barrier to reach the brain where the action of the inhibitor takes place when used to treat cognitive disorders. This is an important feature for the compounds to be good candidates for use in the therapy of cognitive disorders.